The present invention relates to analytical chemistry and, more particularly, to analyte quantification using a mass biosensor system. A major objective of the present invention is to provide for more accurate biosensor quantifications.
Mass biosensor systems use sensors with a biochemical coating that selectively binds a sample component to the substantial exclusion of other sample components. Monitoring mass change that occurs due to the binding permits quantification of even trace amounts of an analyte, i.e., a component of interest, in a sample. Biosensor applications include protein analysis, blood/urine analysis, and environmental studies requiring that the amounts of various pollutants on land and in water be monitored.
There are contrasting strategies for quantifying a sample analyte using a mass biosensor. In a "direct" strategy, a sensor is coated with a "complement" of the sample analyte. Herein, a "complement" of a chemical selectively binds that chemical. For example, to directly quantify an antigen analyte, a sensor is coated with the complementary antibody. When the sample is introduced to the sensor, sample antigen analyte is bound to the antibody, and thus to the sensor. The initial rate at which analyte binds is directly related to the concentration of the sample analyte. Thus, monitoring the rate of mass change due to this binding permits the sample analyte concentration to be determined.
In an "indirect" approach, the sample analyte is not bound to the sensor. The sensor is prepared with a coating which is an analog of the analyte. An "analog" of an analyte is a chemical entity that behaves the same with respect to a given complement to the analyte as does the analyte itself. An analyte is necessarily an analog of itself, and derivatives of an analyte can be analogs. A prequantified complement to the analyte is added to the sample, which is then introduced to the sensor. The sample analyte competes with the analog coating on the sensor for the complement; thus, a higher analyte concentration corresponds to a slower rate of binding to the sensor. The mass of the complement binding to the sensor is monitored, permitting the concentration of the analyte in the sample to be determined.
To provide a description of mass biosensor operation that is applicable to both the direct and indirect strategies, the term "binder" refers to the selective sensor coating, and the term "ligand" is used to refer to the substance that the selective sensor coating specifically binds. In the direct strategy, the ligand is the analyte and the binder is the complement of the ligand. In the indirect strategy, the ligand is the complement of the analyte, and the binder is the analog of the analyte. The binding between the ligand and binder is "specific" in that it occurs between respective specific molecular locations on the ligand and binder. Mechanisms for specific binding can include electrostatic binding, hydrogen bonds, and van der Waals (hydrophobic) binding.
One type of mass biosensor uses a piezoelectric crystal as an acoustic waveguide. An input transducer generates a periodic acoustic signal from a periodic electrical input signal. The acoustic signal propagates through the crystal to an output transducer that converts the received acoustic signal to an electrical output signal. The propagation velocity of the acoustic signal changes as the mass of ligand bound to the surface of the crystal changes.
In one sensor system configuration, a "sample" signal passes through a "sample" sensor, and an identically generated "reference" signal is passed through a "reference" sensor. The reference and sample sensors are matched, except for the affinities of the coatings. While the sample sensor coating has an affinity for the ligand of interest, the reference coating does not specifically bind that ligand. Preferably, the reference coating is selected to match the molecular mass and other binder properties, with the exception of ligand affinity.
When sample is introduced to the sample sensor, ligand binds to the binder, increasing the mass attached to the sensor, and thus, the propagation velocity of the sample signal changes. The change in propagation velocity is reflected in a change of phase in the sample signal. The phase changes can be monitored by comparing the sample signal phase with that of the reference signal. Initially, the sample signal phase increases in proportion to the concentration of the ligand in the sample, exclusive in the indirect approach of ligand bound to analyte. As binding sites become occupied, the binding rate decreases. If sufficient time is provided for an equilibrium to be reached between binding and dissociation, the net binding rate falls to zero; the mass at equilibrium can be used to calculate the analyte concentration. However, the initial mass change rate, as indicated by the initial phase change rate, provides for the determination of analyte concentration before and whether or not equilibrium is achieved.
As is apparent from the foregoing, there is no requirement that mass be determined explicitly for concentration to be determined. Accordingly, "mass measurement" and "monitoring mass" encompass detecting and monitoring variables that vary with mass. As explained above, the primary variable used to determine analyte concentration is the initial phase change rate provided by a mass biosensor system when a sample is introduced to the incorporated sample sensor. Alternative variables are equilibrium phase offset, frequency, explicitly determined mass or mass change rate, and other mass-sensitive variables associated with other types of mass biosensor systems.
The algorithm used to calculate analyte concentration in a sample depends on the measurement strategy employed. In the direct approach, the analyte is bound to the sensor so that analyte concentration is directly related to the initial phase change rate and the affinity binding constant for ligand analyte and the binder sensor coating. In the indirect approach, the ligand is the complement and its concentration is predetermined. The sample analyte and the analog binder compete for the predetermined quantity of ligand. A greater quantity of analyte more effectively competes for the ligand complement, and results in a lower initial phase change rate. The algorithm used in the indirect approach reflects this competition. In either approach, the appropriate algorithm preferably involves comparing measurement results with one or more calibration points determined from "standard" calibration measurements using known quantities of analyte.
The accuracy of a biosensor measurement is limited by the repeatability of the measurement process. Ideally, two measurements of the same sample yield the same measurements, and the differences between measurements reflect the differences between samples to the exclusion of artifacts. To the extent that ambient conditions and sensor variations contribute to measurement differences, measurement accuracy is impaired. This impairment applies generally to comparisons between measurements, and, more specifically, between a sample measurement and one or more blank measurements used to calibrate measurements. A lower repeatability impairs the validity of the calibration, and, thus, the accuracy of the measurement.
The repeatability of a biosensor method is affected by the nature of the bond attaching the binder to the substrate. Binder can be covalently attached or nonspecifically adsorbed to an underlying substrate, either directly or through an intermediate structure. Adsorption results in relatively weak attachment of binder molecules in random, unknown, and/or unstable orientations. Weakly attached binder can become dissociated during or between measurements, impairing repeatability. Some orientations can prevent ligands from reaching a binding site on a binder molecule. Finally, the relative instability of adsorbed binder can subject the binder to indeterminate effects due to the action of sample components, some of which may be unknown or uncharacterized. All of these disadvantages limit the repeatability of measurements using these sensors and subject measurements to systematic distortions due to differences in the coatings between the time of calibration and the time of measurement.
More recently, techniques have been developed for covalently binding a wide variety of ligand binders to the substrate of a sensor of a mass biosensor system. Covalent bonds are relatively stable and provide the binder with a predictable orientation that can ensure the accessibility of the binding sites required for ligand binding.
Covalently binding sensor coatings is relatively tedious and costly, raising the cost of individual sensors and discouraging end users from reusing sensors. After a prepared crystalline surface is used for measurement, many of its ligand-binding sites are occupied by ligands. Accordingly, the sensor is not suitable for use in measuring the quantity of the same ligand in a different sample solution. In practice, a new sensor is required for each "run", whether the run is for calibration or measurement. Differences between a sensor used in a calibration measurement and one used in a sample measurement result in systematic errors, which impair accuracy.
The need to use different sensors for calibration measurements and sample measurements can be avoided by effective renewal of a sensor. Theoretically, a sensor can be renewed by removing the binder and ligand, then reapplying a binder coating. In practice, it is difficult to remove a covalently bound binder. In addition, covalently re-attaching binder coatings is a critical and expensive operation. Once again, systematic errors loom, since the second coating can differ significantly from the first. It is not feasible for the end user to reapply a covalent binder coating between a calibration run and a measurement. Thus, accuracy is limited where replacement of sensor coatings is required.
Preferably, renewal would be effected by removing ligand without disturbing the binder coating. In this case, the same substrate and coating would be used in both a calibration run and a measurement, minimizing systematic errors. A dissociating treatment can be applied to split specific ligand-to-binder bonds. Care must be taken to remove virtually all the ligand to avoid biasing the succeeding run. However, too strong an application of a dissociation reagent can degrade the binder and even separate it from the crystalline surface.
In theory, the amount of dissociation reagent could be predetermined for each ligand. However, the repeatability of dissociation reagents is imperfect. Variations in ligand conjugates, dissociation reagent concentrations, flow configurations, ambient conditions, and other chemicals in solution can lead to under or over dissociation.
Renewal of sensors with adsorbed binder is less of a problem. While it is difficult to remove attached ligand from the binder without impairing the latter, it is practical to remove the binder along with any attached ligand and then adsorb fresh binder. The problem remains that the new coating can differ randomly from the original.
What is needed is a more accurate mass biosensor method. Such a method should minimize systematic distortions between calibration runs and sample analyses. Moreover, such method should provide for sensor coatings with the stability and predictable orientation characterizing sensors with covalently bound coatings.